World-wide, spurious thrombosis accounts for 1 in 4 non-communicable disease deaths. Normally platelets respond to vascular damage and secrete granule cargo that are essential for recruiting more platelets, their adhesion, and thrombosis. This releasate promotes normal sequelae but can also contribute to disease. Do platelets actively control what they release? How much releasate is enough? These are key questions to understand how platelets affect their vascular microenvironments. Membrane proteins (v- and t-SNAREs) fuse granule and plasma membranes to allow cargo release. In platelets, VAMPs, SNAP-23, and Syntaxin-11 form a membrane-spanning complex that mediates fusion. Formation of this complex requires post-translational modifications and a host of SNARE-regulators. This controls the complexity of the platelet release reaction: its rate, extent, and content. We hypothesize that by manipulating the platelet secretory machinery, we can modulate thrombosis with only modest effects on hemostasis. We propose three Aims: 1) Determine how alterations in platelet exocytosis affect thrombosis and hemostasis. Using our unique collection of transgenic mice, we will genetically titrate platelet secretory machinery to determine how much secretion is needed for thrombosis and hemostasis. 2) Determine if platelet exocytosis can be controlled by targeting post-translational modifications of t-SNAREs. We will examine two post-translational modifications: SNAP-23 phosphorylation, by I?B kinase, and t-SNARE acylation. We will test inhibitors to determine if they can be repurposed as anti-thrombotics. t- SNARE acylation is highly dynamic in platelets. We will alter its cycling with acyl transferase and thioesterase inhibitors to define its importance to secretion and whether acylation can be targeted to affect platelet exocytosis. 3) Determine the roles of new elements of the platelet exocytosis machinery. We will probe the roles of two novel secretory elements: ?-synuclein and RalA/B. Our proposal expands our knowledge of the molecular requirements and the sequence of protein-protein interactions controlling platelet exocytosis. Our work is significant because it provides the needed mechanistic insights to identify potential targets for therapeutic intervention and to evaluate the relevance of the increasing volume of gene/risk associations.